The present invention relates to novel uses of Glial-cell Derived Neurotrophic Factor (xe2x80x9cGDNFxe2x80x9d) and its receptor designated GDNFRxcex1, and provides GDNFRxcex1-encoding nucleic acid (SEQ ID NO:1) and amino acid sequences (SEQ ID NO:2). In particular, the invention relates to native sequence GDNFRxcex1, GDNFRxcex1 variants, soluble GDNFRxcex1 variants including GDNFRxcex1 extracellular domain, chimeric GDNFRxcex1, and antibodies which bind to the GDNFRxcex1 diagnostic techniques for identifying GDNF-related conditions, methods for identifying molecules homologous to GDNFRxcex1, and therapeutic techniques for the treatment of GDNF-related and GDNFRxcex1-related conditions, particularly kidney diseases.
Diseases of the nervous system are usually devastating and often lead to death. Neurological diseases are often chronic, which imposes a great social and economic burden. For example, stroke is the third leading cause of death in the United States, after heart disease and cancer. Neurotrophic factors, which are naturally-occurring proteins, such as insulin-like growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, -4/5 and- 6, ciliary neurotrophic factor, GDNF, and recently neurturin, have been proposed as potential means for enhancing specific neuronal cell survival to treat neurological diseases such as amyotrophic lateral sclerosis, Alzheimer""s disease, stroke, epilepsy, Huntington""s disease, Parkinson""s disease, and peripheral neuropathy. Neurotrophic factors, or neurotrophins, which influence growth and development of the vertebrate nervous system, are believed to play an important role in promoting the differentiation, survival, and function of diverse groups of neurons in the brain and periphery. Neurotrophic factors are believed to have important signaling functions in neural tissues, based in part upon the precedent established with nerve growth factor (NGF). NGF supports the survival of sympathetic, sensory, and basal forebrain neurons both in vitro and in vivo. Administration of exogenous NGF rescues neurons from cell death during development. Conversely, removal or sequestration of endogenous NGF by administration of anti-NGF antibodies promotes such cell death (Heumann, J. Exp. Biol., 132:133-150 (1987); Hefti, J. Neurosci., 6:2155-2162 (1986); Thoenen et al., Annu. Rev. Physiol, 60:284-335 (1980)).
Additional neurotrophic factors related to NGF have since been identified. These include brain-derived neurotrophic factor (BDNF) (Leibrock, et al., Nature, 341:149-152 (1989)), neurotrophin-3 (NT-3) (Kaisho, et al, FEBS Lett., 266:187 (1990); Maisonpierre, et al., Science, 247:1446 (1990); Rosenthal, et al., Neuron, 4:767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berkmeier, et al., Neuron, 7:857-866 (1991)). GDNF, a distant member of the TGF-xcex2 super family, and neurturin (xe2x80x9cNTNxe2x80x9d) are two, recently identified, structurally related, potent survival factors for sympathetic sensory and central nervous system neurons (Lin et al. Science 260:1130-1132 (1993); Henderson et al. Science 266:1062-1064 (1994); Buj-Bello et al., Neuron 15:821-828 (1995); Kotzbauer et al. Nature 384:467-470 (1996)). GDNF has been considered a potential therapeutic agent for Parkinson""s disease, ALS, and Alzheimer""s disease. The mechanism by which GDNF and NTN signals are transmitted has not been elucidated.
Neurotrophins, like NGF, affect their target cells through interactions with cell surface receptors. According to our current understanding, two kinds of transmembrane glycoproteins act as receptors for the known neurotrophins. Equilibrium binding studies have shown that neurotrophin-responsive neuronal cells possess a common low molecular weight (65,000-80,000 Daltons), a low affinity receptor typically referred to as p75LNGFR or p75, and a high molecular weight (130,000-150,000 Dalton) receptor. The high affinity receptors (trkA, trkB and trkC) are members of the trk family of receptor tyrosine kinases.
Receptor tyrosine kinases are known to serve as receptors for a variety of protein factors that promote cellular proliferation, differentiation, and survival. In addition to the trk receptors, examples of other receptor tyrosine kinases include the receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). Typically, these receptors span the cell membrane, with one portion of the receptor being intracellular and in contact with the cytoplasm, and another portion of the receptor being extracellular. Binding of a ligand to the extracellular portion of the receptor induces tyrosine kinase activity in the intracellular portion of the receptor, with ensuing phosphorylation of various intracellular proteins involved in cellular signaling pathways.
Aberrant expression of receptor tyrosine kinases (xe2x80x9cRTKxe2x80x9d) correlates with transforming ability. For example, carcinomas of the liver, lung, breast and colon show elevated expression of Eph RTK. Unlike many other tyrosine kinases, this elevated expression can occur in the absence of gene amplification or rearrangement Moreover, Hek, a human RTK, has been identified as a leukemia-specific marker present on the surface of a pre-B cell leukemia cell line. As with Eph, Hek also was overexpressed in the absence of gene amplification or rearrangements in, for example, hemopoietic tumors and lymphoid tumor cell lines. Over-expression of Myk-1 (a murine homolog of human Htk (Bennett et al., J. Biol. Chem., 269(19): 14211-8 (1994)) was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene. (Andres et al., Oncogene, 9(5):1461-7 (1994) and Andres et al., Oncogene, 9(8):2431 (1994)). Ret, the product of the c-ret proto-oncogene, is a member of the receptor tyrosine kinase superfamily.
In addition to their roles in carcinogenesis, a number of transmembrane tyrosine kinases have been reported to play key roles during development. Some receptor tyrosine kinases are developmentally regulated and predominantly expressed in embryonic tissues. Examples include Cek1, which belongs to the FGF subclass, and the Cek4 and Cek5 tyrosine kinases (Pasquale et al., Proc. Natl. Acad. Sci., USA, 86:5449-5453 (1989); Sajjadi et al., New Biol., 3(8):769-78 (1991); and Pasquale, Cell Regulation, 2:523-534 (1991). Eph family members are expressed in many different adult tissues, with several family members expressed in the nervous system or specifically in neurons (Maisonpierre et al., Oncogene, 8:3277-3288 (1993); Lai et al., Neuron, 6:691-704 (1991)).
The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result in different malignancies and pathological disorders. Therefore, there exists a need to identify means to regulate, control and manipulate receptor tyrosine kinases (xe2x80x9cRTKxe2x80x9d) and their associated ligands or GPI-linked receptors, in order to provide new and additional means for the diagnosis and therapy of receptor tyrosine kinase pathway-related disorders and cellular processes. The present application provides the clinician and researcher with such means by providing a novel neurotrophin-binding molecule that is also specific for interacting with a particular RTK receptor. New disease conditions are identified that are associated with this molecule and its neurotrophin ligand. These compounds and their methods of use, as provided herein, allow new and exquisite therapeutic control and specificity. Accordingly, it is an object of the present invention to provide an improved therapy for the prevention and/or treatment of neurological conditions and other conditions in which neurotrophic signaling pathways related to this novel receptor and its ligand play a role.
These and other objects of the invention will be apparent to the ordinarily skilled artisan upon consideration of the specification as a whole.
The present invention is based, in part, on the present discovery that GDNF-deficient mice completely lack kidneys and enteric nervous system and display a partial loss of dorsal root ganglia ( less than 23%) and sympathetic ( less than 35%) and nodose sensory ganglia ( less than 40%) neurons. GDNF-heterozygotes display severe end stage renal disease at an early age. Thus GDNF plays an essential role in the development or survival of the metanephric kidney and enteric neurons. Accordingly, provided are methods of treatment of these and related diseases using GDNF and GDNF-like compounds, optionally in complex or combination with GDNF receptor.
Provided herein are a novel GDNF receptor termed GDNFRxcex1, soluble forms of the receptor, and a GDNFRxcex1 extracellular domain (xe2x80x9cECDxe2x80x9d). Also disclosed are GDNFRxcex1 polypeptides, optionally conjugated with or fused to molecules which increase the serum half-lives thereof, and optionally formulated as pharmaceutical compositions with a physiologically acceptable carrier.
Soluble GDNFRxcex1 that retains both ligand binding, preferably GDNF binding, and receptor signaling function (via Ret receptor tyrosine kinase) can be used to impart, restore, or enhance GDNFRxcex1-ligand (preferably GDNF) responsiveness to cells. This responsiveness includes ligand-binding, Ret tyrosine phosphorylation and Ret-mediated downstream activity, which can result in modulation of cell activity such as survival or growth. The embodiments find use in vivo, in vitro or ex vivo. The compounds of the invention find use in treating conditions known to be associated with GDNF as well as the newly identified conditions disclosed herein. GDNFRxcex1 ECD that binds GDNF, but does not mediate a GDNF signal, can be used as an antagonist to sequester GDNF ligand to reduce activation of endogenous GDNFRxcex1. This is useful in conditions characterized by excess levels of GDNF ligand and/or excess GDNFRxcex1 activation in a mammal.
Pharmaceutical compositions of soluble GDNFRxcex1, preferably ECD, further include an GDNFRxcex1 ligand, preferably GDNF. Such compositions, comprising a ligand/GDNFRxcex1 complex, are useful where it is desirable to prolong the half-life of the ligand, provide slow, sustained release of ligand, impart GDNFRxcex1-ligand responsiveness to a target cell, and/or activate or enhance endogenous cellular GDNFRxcex1 or Ret activity directly. Optionally, the composition further contains one or more cytokines, neurotrophic factors, or their agonist antibodies.
Chimeric GDNFRxcex1 molecules such as GDNFRxcex1 immunoadhesins (having long serum half-lives) and epitope-tagged GDNFRxcex1 are disclosed. These find particular use as soluble forms of GDNFRxcex1, particularly in complexes to deliver GDNF or impart GDNF-responsiveness to cells. Bispecific immunoadhesins (for example, combining a GDNFRxcex1-ligand binding activity with a ligand-binding domain of another cytokine or neurotrophic factor receptor) can form high affinity binding complexes for GDNFRxcex1-ligands in combination with other factors or for targeted delivery.
Also provided are methods for identifying a molecule which binds to and/or activates GDNFRxcex1. Thus assays are provided to screen for or identify GDNFRxcex1-ligand molecules (such as peptides, antibodies, and small molecules) that are agonists or antagonists of GDNFRxcex1. Such methods generally involve exposing an immobilized GDNFRxcex1 to a molecule suspected of binding thereto and determining binding of the molecule to the immobilized GDNFRxcex1 and/or evaluating whether or not the molecule activates (or blocks activation of) the GDNFRxcex1. In order to identify such GDNF ligands, the GDNFRxcex1 can be expressed on the surface of a cell and used to screen libraries of synthetic candidate compounds or naturally-occurring compounds (e.g., from endogenous sources such as serum or cells). GDNFRxcex1 can also be used as a diagnostic tool for measuring serum levels of endogenous or exogenous GDNFRxcex1-ligand.
In a further embodiment, a method for purifying an GDNFRxcex1-ligand is provided. This finds use in commercial production and purification of therapeutically active molecules that bind to this receptor. In one embodiment the molecule of interest (generally in a composition comprising one or more contaminants) is adsorbed to immobilized GDNFRxcex1 (e.g. GDNFRxcex1 immunoadhesin immobilized on a protein A resin). The contaminants, by virtue of their inability to bind to the GDNFRxcex1, will generally not bind the resin. Accordingly, it is then possible to recover the molecule of interest from the resin by changing the elution conditions, such that the ligand molecule is released from the immobilized receptor.
Antibodies are provided that specifically bind to GDNFRxcex1. Preferred antibodies are monoclonal antibodies that are non-immunogenic in a human and bind to an epitope in the extracellular domain of the receptor. Preferred antibodies bind the GDNFRxcex1 with an affinity of at least about 106 L/mole, more preferably 107 L/mole. Preferred antibodies are agonist antibodies.
Antibodies, which bind to GDNFRxcex1, can be optionally fused to a heterologous polypeptide. The antibody or fusion finds particular use to isolate and purify GDNFRxcex1 from a source of the receptor.
In a further aspect is provided a method for detecting GDNFRxcex1 in vitro or in vivo which includes the steps of contacting an GDNFRxcex1 antibody with a sample suspected of containing the receptor, and detecting if binding has occurred.
For certain applications it is desirable to have an agonist antibody. Such agonist antibodies are useful for activating GDNFRxcex1 as described for GDNFRxcex1-ligands such as GDNF. Furthermore, these antibodies are useful to treat conditions in which an effective amount of GDNFRxcex1 activation leads to a therapeutic benefit in the mammal. For example, the agonist antibody can be used to elicit an GDNF response in a cell comprising GDNFRxcex1 and, preferably, Ret. For therapeutic applications it is desirable to prepare a composition having the agonist antibody and a physiologically acceptable carrier. Optionally, the composition further contains one or more cytokines, neurotrophic factors, or their agonist antibodies.
In other embodiments, the antibody is a neutralizing antibody. Such molecules can be used to treat conditions characterized by unwanted or excessive activation of GDNFRxcex1.
In addition to the above, the invention provides isolated nucleic acid molecules, expression vectors and host cells encoding GDNFRxcex1, GDNF, or agonist thereof, which can be used in the recombinant production of GDNFRxcex1, GDNF, or agonist thereof, as described herein. The isolated nucleic acid molecules and vectors are also useful to prepare transgenic animals, for gene therapy applications to treat patients with defects in GDNFRxcex1 or GDNF, to increase responsiveness of cells to GDNFRxcex1 ligands, or alternatively to decrease GDNFRxcex1 or GDNF activity (as by use of antisense nucleic acid).